TNIP1 Regulates Myddosome Dynamics During IL-1β Signaling

Abstract

Entzündungen sind ein mehrstufiger Prozess des Immunsystems von Säugetieren zur Aufrechterhaltung der Homöostase, der durch das komplexe Zusammenspiel verschiedener Zytokine wie Interleukin 1β (IL-1β) vermittelt wird. Die IL-1β-Signaltransduktion ist für die akute Entzündung von entscheidender Bedeutung, muss aber gleichzeitig streng reguliert werden, da eine veränderte IL-1β-Signalübertragung entweder zu einer Hypo- oder Hyperinflammation führt. Die Bindung von IL-1β an seinen Rezeptor induziert die sequentielle Rekrutierung des myeloiden Differenzierungsgenes 88 (MyD88) und der IL-1-Rezeptor-assoziierten Kinasen (IRAKs) 4 und 1. Zusammen bilden sie einen makromolekularen Komplex, das Myddosom. Daraufhin aktiviert das Myddosom den NF-κB- und den MAPK-Signalweg. Diese Aktivierung wird durch die räumliche und zeitliche Dynamik der nachgeschalteten Signalwegnetze kodiert. Wie setzt das intrazelluläre IL-1β-Signalnetzwerk den extrazellulären Nachweis von IL-1β effizient in eine präzise und angemessene zelluläre Reaktion um? Welche Kontrollmechanismen kommen zum Einsatz, um eine angemessene Antwort zu gewährleisten und eine Hypo- oder Hyperantwort zu verhindern?

Diese Arbeit charakterisiert die IL-1β-vermittelte Signalwegdynamik mithilfe der Immunpräzipitations-Massenspektrometrie (IP-MS). Dazu verwendete ich endogen gen-editierte EL4- Zelllinien mit Fluorophorproteinen an allen Myddosom-Proteinen MyD88, IRAK4 und IRAK1. Mittels statistischer Analysen identifizierte ich das Interaktom dieser Proteine nach 15-, 30- und 60-minütiger IL-1β-Stimulation und untersuchte, wie sich dieses Interaktom im Laufe der Zeit veränderte. Anschließend identifizierte ich mithilfe von Signalwegannotationsanalysen Proteine, die potentiell an der Runterregulierung des IL-1β-Signalwegs beteiligt sind.

Um zu verstehen, wie das IL-1β-Signalwegnetzwerk die Translationsmaschinerie in EL4 Zellen beeinflusst, um eine angemessene Reaktion zu gewährleisten, untersuchte ich außerdem den IL-1β-abhängigen Proteinumsatz. Konkret wandte ich gepulste stabile Isotopenmarkierung durch Aminosäuren in der Zellkultur (pSILAC) in Kombination mit Azidohomoalanin (AHA)- Klickchemie und MS nach 30-, 60-, 120- und 240-minütiger IL-1β-Stimulation an

Das Ergebnis dieser Proteomik-Untersuchungen war die Identifizierung des TNFα-induzierten Proteins 3 (Tnfaip3) interagierendes Protein 1 (TNIP1) als potenziellen Kandidaten für die Herunterregulierung des IL-1β-Signalwegs. Nach IL-1β-Stimulation kolokalisiert TNIP1 mit allen Myddosomen-Proteinen sowie mit Tnfaip3. Diese Deubiquitinase wird ebenfalls zum Myddosom rekrutiert. Mittels CRISPR/Cas9 erzeugte ich eine TNIP1-KO-EL4 Zelllinie. Nach IL-1β-Stimulation zeigten TNIP1-KO-Zellen vermehrt phosphoryliertes p65, aber verringertes phosphoryliertes JNK sowie eine langfristig verringerte IL-2-Sekretion. Daher ist TNIP1 nicht nur an der Herunterregulierung des NF-κB-Signalwegs beteiligt, sondern aktiviert auch den MAPK-Signalweg.

Alternate abstract:

Inflammation is a multistep process of the mammalian immune system to maintain homeostasis, mediated by the complex interplay of different cytokines such as interleukin 1β (IL-1β). IL-1β signal transduction is crucial for acute inflammation, but at the same time needs tight regulation as altered IL-1β signaling results in either hypo- or hyperinflammation. IL-1β binding to its receptor induces the recruitment of myeloid differentiation primary response gene 88 (MyD88), IL-1 receptor associated kinases (IRAKs) 4 and 1 sequentially, resulting in a macromolecular complex, called the myddosome. The formed myddosome triggers activation of NF-κB signaling and the MAPK pathways. This response is encoded by the spatial and temporal dynamics of downstream signaling networks. How does the intracellular IL-1β signaling network efficiently convert the extracellular detection of IL-1β into a precise and proportionate cellular response? What control mechanisms apply in order to ensure a proportionate response and prevent a hypo- or hyper response?

This study characterizes the IL-1β mediated signaling dynamics using immunoprecipitation purification mass spectrometry (IP-MS). I used endogenously gene-edited EL4 cell lines that expressed myddosome proteins MyD88, IRAK4 and IRAK1 tagged with genetically encoded fluorescent proteins. Using statistical analysis, I identified the interactome of these proteins, and assayed how this interactome remodeled in time by comparing interactomes generated after 15-, 30- and 60-min of IL-1β stimulation. Next, I identified proteins potentially involved in IL1β signaling downregulation using pathway annotation analysis.

Further, in order to understand how the IL-1β signaling network affects the translational machinery to ensure a proportionate response, I investigated IL-1β-dependent protein turnover in EL4 cells. Specifically, I applied pulsed stable isotope labeling by amino acids in the culture (pSILAC) combined with azidohomoalanine (AHA)-click chemistry and MS after 30-, 60-, 120- and 240-min of IL-1β stimulation.

The result of these proteomics approaches was the identification of TNFα induced protein 3 (Tnfaip3) interacting protein 1 (TNIP1) as a potential candidate in IL-1β signal downregulation. TNIP1 co-localizes with all myddosome proteins after IL-1β stimulation. TNIP1 also colocalized with the deubiquitinase Tnfaip3, which was also recruited to myddosomes. I generated a TNIP1 KO EL4 cell line using CRISPR/Cas9. After IL-1β stimulation, TNIP1 KO cells show increased levels of phosphorylated p65, but decreased levels of phosphorylated JNK as well as decreased levels of long-term IL-2 secretion. Therefore, TNIP1 is not only involved in downregulatory NF-κB signaling, but activates MAPK pathway.

Inflammation is a multistep process of the mammalian immune system to maintain homeostasis, mediated by the complex interplay of different cytokines such as interleukin 1β (IL-1β). IL-1β signal transduction is crucial for acute inflammation, but at the same time needs tight regulation as altered IL-1β signaling results in either hypo- or hyperinflammation. IL-1β binding to its receptor induces the recruitment of myeloid differentiation primary response gene 88 (MyD88), IL-1 receptor associated kinases (IRAKs) 4 and 1 sequentially, resulting in a macromolecular complex, called the myddosome. The formed myddosome triggers activation of NF-κB signaling and the MAPK pathways. This response is encoded by the spatial and temporal dynamics of downstream signaling networks. How does the intracellular IL-1β signaling network efficiently convert the extracellular detection of IL-1β into a precise and proportionate cellular response? What control mechanisms apply in order to ensure a proportionate response and prevent a hypo- or hyper response?

This study characterizes the IL-1β mediated signaling dynamics using immunoprecipitation purification mass spectrometry (IP-MS). I used endogenously gene-edited EL4 cell lines that expressed myddosome proteins MyD88, IRAK4 and IRAK1 tagged with genetically encoded fluorescent proteins. Using statistical analysis, I identified the interactome of these proteins, and assayed how this interactome remodeled in time by comparing interactomes generated after 15-, 30- and 60-min of IL-1β stimulation. Next, I identified proteins potentially involved in IL1β signaling downregulation using pathway annotation analysis.

Further, in order to understand how the IL-1β signaling network affects the translational machinery to ensure a proportionate response, I investigated IL-1β-dependent protein turnover in EL4 cells. Specifically, I applied pulsed stable isotope labeling by amino acids in the culture (pSILAC) combined with azidohomoalanine (AHA)-click chemistry and MS after 30-, 60-, 120- and 240-min of IL-1β stimulation.

The result of these proteomics approaches was the identification of TNFα induced protein 3 (Tnfaip3) interacting protein 1 (TNIP1) as a potential candidate in IL-1β signal downregulation. TNIP1 co-localizes with all myddosome proteins after IL-1β stimulation. TNIP1 also colocalized with the deubiquitinase Tnfaip3, which was also recruited to myddosomes. I generated a TNIP1 KO EL4 cell line using CRISPR/Cas9. After IL-1β stimulation, TNIP1 KO cells show increased levels of phosphorylated p65, but decreased levels of phosphorylated JNK as well as decreased levels of long-term IL-2 secretion. Therefore, TNIP1 is not only involved in downregulatory NF-κB signaling, but activates MAPK pathway.